The D1 dopamine receptor (D1R) has been implicated in numerous neuropsychiatric disorders and various D1R-selective ligands have shown potential as therapeutic agents. In an effort to identify novel selective allosteric modulators of the D1R we used a high throughput screening approach. 380,000 small molecules in the NIH Molecular Libraries Screening Center Network (MLPCN) library were screened using a cell line expressing the D1R coupled to G15 resulting in a robust Ca2+ signal upon receptor stimulation. Hit compounds were triaged through secondary functional and radioligand displacement binding assays to determine subtype selectivity and their allosteric versus orthosteric nature. We initially identified 96 putative positive allosteric modulator (PAM) hits that enhanced the EC20 activity of dopamine in the Ca2+ response and 6 of these were subsequently confirmed during triage and were chosen for further characterization. In addition we found approximately 115 agonist and 125 antagonist hits that failed to completely inhibit radioligand binding and thus have been classified as potential allosteric agonists and antagonists or negative allosteric modulators (NAMs). These compounds are currently being characterized using additional assays to confirm their selectivity, activity, and classification. Four of the PAM hits have selected for chemical optimization, which is ongoing now. The D2 dopamine receptor (D2R) is also involved in the etiology and/or therapy of many neuropsychiatric disorders. Unfortunately, truly specific drugs for this receptor have been difficult to obtain, primarily due to high conservation of the orthosteric binding site within dopamine receptor (DAR) subtypes and other G protein-coupled receptors (GPCRs). We have employed a high throughput-screening approach using the 380,000 small molecule NIH MLPCN library to identify novel allosteric modulators of the D2R. The primary screen was conducted using a cell line expressing an inducible D2R coupled to a chimeric Gqi5 protein, thus linking receptor activation to Ca2+ mobilization. Hits were subjected to an extensive triage strategy to characterize DAR activity and selectivity. On the basis of these analyses, 745 agonist and 499 antagonist compounds were selected and evaluated using radioligand binding competition assays to identify the nature of their receptor interactions (orthosteric or allosteric). Compounds that are ineffective in competing for binding likely exert their functional activity via allosteric mechanisms. These experiments resulted in the identification of 47 agonists and 48 antagonists that had insignificant effects on radioligand binding when tested at concentrations up to 40 uM, despite exhibiting maximal functional effects at significantly lower concentrations. These compounds would thus appear to be allosteric agonists and negative allosteric modulators of the D2R. Further characterization is ongoing. The D2R can activate a spectrum of signaling cascades primarily through G proteins and beta-arrestin recruitment, making it an attractive target for the development of signaling biased ligands. Unlike dopamine, which simultaneously activates G proteins and recruits beta-arrestins, a biased ligand affects only one pathway, and the development of such ligands can allow for a more fine-tuned study of receptor signaling. Our lab has identified a compound (ML1547) that is a highly efficacious agonist at D2R-mediated G protein-linked signaling, but it does not recruit beta-arrestin. Rather, this compound is an antagonist of D2R-stimulated beta-arrestin-mediated signaling. A number of structural analogs of ML1547 were characterized for their signaling properties, which ranged from fully biased, partially biased, to unbiased. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally-selective properties of these compounds. This, along with medicinal chemistry approaches, will allow for the identification of more potent G protein-biased analogs of ML1547 that will enable a highly targeted approach for investigating D2R signaling in vivo, and may eventually allow for selective treatment of disease symptoms associated with D2R dysfunction. Many currently available dopaminergic drugs modulate both D2 and D3 DARs due to high homology in their orthosteric binding sites, leading to potential unwanted side effects, but also uncertainty as to the roles that each DAR subtype plays in normal and pathological processes. In order to discover compounds that target unique, less conserved allosteric sites of these DARs, our lab employed a high throughput screening approach. Through the NIH Molecular Libraries Program, compound 3843 was originally identified as a D2R antagonist in a screen of a 380,000+ small molecule library. Counter-screening assays of beta-arrestin recruitment revealed that this compound selectively activates the D3 DAR (D3R), yet also acts as an antagonist at the D2R. Over 270 analogs were synthesized and tested to explore the structure-activity relationship of 3843 for the D2R and D3R, and several compounds with a 1,000-fold or greater increase in D3R agonist potency were found. Initial competition binding assays suggest these compounds act in an allosteric manner at the D3R, and initial ADME studies performed on 3843 were favorable. We ultimately hope these probes will be useful as vitro and in vivo pharmacological tools to elucidate D3R specific physiology and pathology. The Chinese herbal extract (-)-stepholidine has been reported to exhibit D2R antagonism and D1R agonist-like activities in a number of in vivo behavioral analyses. On this basis, it has been proposed as a potential novel antipsychotic agent. Prior studies, however, have shown complicated and mixed pharmacological responses involving both dopamine and serotonin receptors. Importantly, clear in vitro characterization of (-)-stepholidnine's actions on individual DAR subtype signaling pathways has not been extensively explored. These data are particularly important given the recent discovery of the potential for signaling bias within the DAR family. In this study we investigated the biological actions and binding activities of (-)-stepholidine on all DAR subtypes. (-)-Stepholidine demonstrated relatively high binding affinity for all DAR subtypes, but failed to display any agonist response in either G protein- (cAMP assays and GTPgS binding), or beta-arrestin (beta-arrestin translocation assays)-mediated signaling at any DAR tested. Furthermore, (-)-stepholidine was a potent full antagonist of dopamine-mediated signaling of both D1- and D2-like receptors and did not display signaling bias, thereby demonstrating that it acts as a non-selective pan-dopamine receptor antagonist. Our conclusion is that (-)-stepholidine lacks agonist activity at D1Rs, as previously proposed, and does not exhibit functional selectivity for D2R signaling.